Prokaryotic diacylglycerol kinase (DAGK) is a 13 kDa integral membrane protein which function as a trimer having 9 transmembrane segments. DAGK plays an essential role in the physiology of many bacteria and is a potential target for anti-microbial agents. DAGK appears to be a structurally unique kinase, but may represent an entire family of uncharacterized membrane proteins. DAGK is an ideal model system for mechanistic studies of membrane-integral biocatalysts. DAGK is a stable protein once correctly folded, but exhibits a tendency to misfold into kinetically-trapped conformations. It may therefore serve as an ideal system in which to examine the relationships of membrane protein folding and misfolding, a matter of pressing medical relevance. The specific aims of the upcoming phase of this project are: Aim 1. Complete determination of the backbone structure of DAGK at moderately high resolution using a combined solution NMR and disulfide mapping approach. Structural information will be critical to illuminate results from the other two aims. Aim 2: Initiate characterization of the roles of functionally critical residues of DAGK using steady-state kinetic methods. These mechanistic studies will shed light upon those aspects of DAGK's function which are peculiar to it by virtue of the membrane environment in which it has evolved. Aim 3. Elucidate key features of wild type DAGK's in vitro folding and misfolding pathways. Examine how these pathways have been perturbed for DAGK mutants which exhibit a particularly high propensity for misfolding. Membrane protein misfolding has rarely been examined under in vitro conditions, despite the relevance of this phenomenon to many diseases.